We have shown that the first nonstructural protein in the DEN2 genome, NS1, can trans complement lethal mutations of this protein in the full-length "infectious" cDNA clone. We have introduced a series of deletions in the carboxy-terminus of the NS1 gene, and in the amino terminus of NS2A in the context of the DEN2 infectious cDNA. NS1 provided in trans can complement several of the carbox-terminal deletions, however one deletion mutant which contains a second, single amino acid deletion in the amino terminus of NS2A, and a twenty five amino acid deletion which includes the entire region under study are not complemented by wildtype NS1. The additional mutation in NS2A does not affect protein cleavage, or dimerization of NS1. We are currently studying the role of NS2A and how it may affect NS1 function. We have also demonstrated that LLC-MK2 cells expressing dengue 2 NS1 show enhanced replication of wildtype dengue 2 by 50 - 100 fold. Initial studies on the mechanism of NS1 enhanced viral replication have shown that control LLC-MK2 cells are sensitive to apoptotic cell death after infection with dengue virus. In contrast, NS1 expressing cells are resistant to virus induced apoptosis. The absence of programmed cell death may allow these cells to produce virus over an extended period of time accounting for enhanced replication and higher titers of virus released. We are currently investigating the role of NS1 in the apoptosis pathway and how it may be affecting cell survival. In parallel, we have analyzed the ability of dengue 2 NS1 espression to enhance replication of the other dengue strains, 1,3, and 4. Comparative studies on virus replication of all dengue stains in control and dengue 2 NS1 expressing cells have shown that dengue 2 NS1 enhances dengue 2 replication 50 - 100 fold; dengue 1 replication 10 - 20 fold; and does not affect the replication of dengue 3 and dengue 4. DNA and protein data support these observations. We are currently analyzing stable cell lines expressing NS1 from each of the dengue strains.